This invention relates to disease resistance in plants.
Grapevine fanleaf virus (GFLV) is a grape nepovirus, which is transmitted from plant to plant by the dagger nematode, Xiphinema index. GFLV is the agent responsible for grapevine fanleaf disease, which occurs worldwide. The disease is named for the fan-leaf shaped appearance of GFLV-infected leaves. It is one of the most damaging and widespread diseases of grapevine. Symptoms of GFLV infection include abnormal shoot morphology and discolorations of the leaves, yielding a fan-like appearance (Agrios, Plant Pathology, 3rd Edition, Academic Press, 1988, pp. 687-688). In addition, fruit production of infected vines is low, with grapevines producing small bunches having abnormal fruit set and ripening. Ultimately, infected grapevines degenerate and die.
Long range spread of GFLV is believed to be by use of infected planting material. While the natural host range is thought to be restricted to grape, GFLV is also transmissible to a wide range of herbaceous species by sap-rubbing inoculation. Chenopodium quinoa is a useful diagnostic species for the virus. In general, GFLV isolates are antigenically uniform and diagnosis by ELISA is a standard procedure.
Current strategies for controlling grapevine fanleaf disease and other nepovirus-induced diseases in vineyards include nematode control (for example, soil fumigation and use of other pesticides), breeding rootstocks for resistance to nematode feeding, breeding grapevines for resistance to GFLV, and planting certified disease-free grapevines.
In general, the invention features a method for producing and selecting a transgenic grapevine or grapevine component having increased resistance to a fanleaf disease. The method generally involves: (a) transforming a grape plant cell with a grape nepovirus coat protein nucleic acid molecule or fragment thereof (for example, a grape nepovirus coat protein nucleic acid molecule or fragment thereof having about 50% or greater sequence identity to SEQ ID NO: 1) which is capable of being expressed in a plant cell; (b) regenerating a transgenic grapevine or grapevine component from the plant cell; and (c) selecting a transgenic grapevine or grapevine component which expresses, at a low level, the nucleic acid molecule or fragment thereof, wherein the low level expression increases the resistance of the transgenic grapevine or grapevine component to fanleaf disease as compared to plants expressing the nucleic acid molecule at a high level. Low level expression of the grape nepovirus mRNA or of the expressed coat protein itself in the transgenic plant is measured according to standard methods including, without limitation, Northern blot analysis, ELISA, and inoculation of transgenic plants with virus and selection of resistant vines. In preferred embodiments, the nucleic acid molecule or fragment thereof is encoded by a transgene found in the transgenic grapevine. In other preferred embodiments, the nucleic acid molecule or fragment thereof is expressed as in a sense or antisense orientation. In yet other preferred embodiments, such grape nepovirus coat protein nucleic acid molecules or fragments thereof (a non-limiting example being a sense nontranslatable grape nepovirus viral coat protein mRNA having an out-of-reading frame initiation ATG initiation codon with the remainder of the mRNA being out of frame) is expressed in the transgenic grapevine or grapevine component.
As is discussed above, the invention also includes fragments of a grape nepovirus coat protein nucleic acid molecule that facilitate, when expressed at low levels, an increased resistance of a transgenic grapevine or grapevine component thereof, to a fanleaf disease. Thus, grape nepovirus coat protein nucleic acid sequences described herein or portions thereof may be expressed in a plant to facilitate disease resistance. Sequences that mediate an increased resistance to a fanleaf disease are considered useful in the invention. As used herein, the term xe2x80x9cfragment,xe2x80x9d as applied to sequences of a nucleic acid molecule, means at least 5 contiguous nucleotides, preferably at least 10 contiguous nucleotides, more preferably at least 20 to 30 contiguous nucleotides, and most preferably at least 40 to 80 or more contiguous nucleotides. Fragments of a grape nepovirus nucleic acid molecule can be produced and, subsequently, integrated into any standard expression vector (for example, those described herein) according to methods known to those skilled in the art.
Preferably, the grapevine useful in the invention is a member of the genus Vitis; and the grapevine component is a somatic embryo, a scion, a rootstock, or a mother block. In still other preferred embodiments, the fanleaf disease is grapevine fanleaf disease caused by a grape nepovirus. In yet other preferred embodiments, the grape nepovirus is a grapevine fanleaf virus or an arabis mosaic virus.
In another aspect, the invention features a vineyard including three or more transgenic grapevines or grapevine components each of which express, at a low level, a grape nepovirus coat protein nucleic acid molecule or fragment thereof, wherein the low level expression of the nucleic acid molecule or fragment thereof increases resistance of the transgenic grapevines or grapevine components in the vineyard to fanleaf disease.
In still another aspect, the invention features a substantially pure protein (for example, a recombinant protein) including an amino acid sequence having at least 97% amino acid identity to the amino acid sequence of the xe2x80x98Genevaxe2x80x99 isolate grape nepovirus coat protein shown in FIGS. 1A-1C (SEQ ID NO: 2). In preferred embodiments, the protein includes the amino acid sequence of the grape nepovirus coat protein shown in FIGS. 1A-1C (SEQ ID NO: 2). In yet other preferred embodiments, the protein has the amino acid sequence of the grape nepovirus coat protein shown in FIGS. 1A-1C (SEQ ID NO: 2) or fragments thereof.
In yet another aspect, the invention features an isolated nucleic acid molecule encoding a protein (for example, a recombinant protein) including an amino acid sequence having at least 97% amino acid identity to the amino acid sequence of the xe2x80x98Genevaxe2x80x99 isolate grape nepovirus coat protein shown in FIGS. 1A-1C (SEQ ID NO: 2). In preferred embodiments, the protein encoded by the nucleic acid molecule includes the amino acid sequence of SEQ ID NO: 2. In yet other preferred embodiments, the protein encoded by the nucleic acid molecule has the amino acid sequence of SEQ ID NO: 2 or a fragment thereof.
In another aspect, the invention features an isolated nucleic acid molecule (for example, a DNA molecule) that encodes a grape nepovirus coat protein that specifically hybridizes to a nucleic acid molecule that includes the nucleic acid sequence of FIGS. 1A-1C (SEQ ID NO: 1). Preferably, the specifically hybridizing nucleic acid molecule encodes a grape nepovirus sequence that mediates resistance when expressed at low levels in a grape plant cell to a fanleaf disease (for example, grapevine fanleaf disease). The invention also features an RNA transcript having a sequence complementary to any of the isolated nucleic acid molecules described above.
In related aspects, the invention further features a cell (for example, a prokaryotic cell or a eukaryotic cell such a mammalian cell or yeast cell) which includes an isolated nucleic acid molecule of the invention. In preferred embodiments, the cell is a bacterium (for example, E. coli or Agrobacterium tumefaciens) or is a plant cell (for example, a grape plant cell from any of the cultivars listed herein). Such a plant cell has resistance against a fanleaf disease (for example, grapevine fanleaf disease).
In still other related aspects, the invention further features a vector (for example, a plant expression vector) which includes an isolated nucleic acid molecule of the invention. In a preferred embodiment, the isolated nucleic acid molecule is operably linked to an expression control region that mediates expression of a protein encoded by the nucleic acid molecule (for example, a nucleic acid molecule (such as DNA) expressed as a sense translatable or a sense nontranslatable mRNA transcript, or as an antisense mRNA transcript).
In still other aspects, the invention features a transgenic plant or plant component (for example, a grapevine or grapevine component) that includes a nucleic acid molecule encoding a protein (for example, a recombinant protein) encoding an amino acid sequence having at least 97% amino acid identity to the amino acid sequence of the xe2x80x98Genevaxe2x80x99 isolate grape nepovirus coat protein shown in FIGS. 1A-1C (SEQ ID NO: 2). In preferred embodiments, such a transgenic plant or plant component includes a nucleic acid molecule of SEQ ID NO: 1. Moreover, fragments of these sequences may be made such that the nucleic acid molecule expresses a sense translatable, sense nontranslatable, or antisense RNA transcript. In still other preferred embodiments, the plant or plant component has the nucleotide sequence of SEQ ID NO: 1 or fragments thereof. Such plants or plant components which include the nucleic acid molecules of the invention have an increased level of resistance against a fanleaf disease caused by a grape nepovirus (for example, GFLV).
The methods and GFLV sequences described herein are useful for providing disease resistance or tolerance or both on a variety of grapevines (for example, Vitis spp., Vitis spp. hybrids, and all members of the subgenera Euvitis and Muscadinia), including scion or rootstock cultivars. Exemplary scion cultivars include, without limitation, those which are referred to as table or raisin grapes and those used in wine production such as Cabernet Franc, Cabernet Sauvignon, Chardonnay (for example, CH 01, CH 02, CH Dijon), Merlot, Pinot Noir (PN, PN Dijon), Semillon, White Riesling, Lambrusco, Thompson Seedless, Autumn Seedless, Niagrara Seedless, and Seval Blanc. Rootstock cultivars that are useful in the invention include, without limitation, Vitis rupestris Constantia, Vitis rupestris St. George, Vitis california, Vitis girdiana, Vitis rotundifolia, Vitis rotundifolia Carlos, Richter 110 (Vitis berlandieri x rupestris), 101-14 Millarder et de Grasset (Vitis riparia x rupestris), Teleki 5C (Vitis berlandieri x riparia), 3309 Courderc (Vitis riparia x rupestris), Riparia Gloire de Montpellier (Vitis riparia), 5BB Teleki (selection Kober, Vitis berlandieri x riparia), SO4 (Vitis berlandieri x rupestris), 41B Millardet (Vitis vinifera x berlandieri), and 039-16 (Vitis vinifera x Muscadinia).
The invention also features scions, rootstocks, somatic or zygotic embryos, cells, or seeds that are produced from any of the transgenic grapevines or grapevine components described herein.
By xe2x80x9cnontranslatablexe2x80x9d is meant an m-RNA sequence that is not translated into a protein. Examples of such nontranslatable sequences include, without limitation, sequences including an initiation ATG codon followed by an engineered frameshift mutation and stop codon to prevent translation of the mRNA into a protein. Grape nepovirus coat protein genes expressing such nontranslatable mRNA sequences may be constructed according to standard methods (for example, those described herein).
By xe2x80x9clow level expressionxe2x80x9d is meant a level of grape nepovirus coat protein gene expression in a transgenic plant that is greater than zero and that is sufficiently low to impart fanleaf disease resistance. xe2x80x9cHigh level expressionxe2x80x9d refers to-the level of gene expression found in a transgenic plant expressing a coat protein gene that is too high to confer resistance to the disease. Exemplary methods for analyzing low level expression of a grape nepovirus coat protein gene includes, without limitation, Northern blot analysis for detection of a mRNA transcript, as well as immunological techniques such as ELISA for detection of a protein.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a protein or nucleic acid molecule exhibiting at least 97%, and preferably 98%, or most preferably 99% identity to a reference amino acid sequence (for example, the amino acid sequence shown in FIGS. 1A-1C; SEQ ID NO: 2) or nucleic acid sequence (for example, the nucleic acid sequences shown in FIGS. 1A-1C; SEQ ID NO: 1). For proteins, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids or greater. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides or greater.
Sequence identity, at the amino acid or nucleic acid levels, is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By a xe2x80x9csubstantially pure proteinxe2x80x9d is meant a grape nepovirus coat protein (for example, the coat protein from the Geneva, N.Y. grape nepovirus isolate (FIGS. 1A-1C; SEQ ID NO: 2)) that has been separated from components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, protein. A substantially pure protein (for example, the coat protein of the xe2x80x98Genevaxe2x80x99 grape nepovirus isolate) may be obtained, for example, by extraction from a natural source (for example, a GFLV-CP infected plant such as C. quinoa); by expression of a recombinant nucleic acid encoding a protein; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By xe2x80x9cisolated nucleic acid moleculexe2x80x9d is meant a nucleic acid molecule (for example, DNA) that is free of the nucleic acids which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the nucleic acid molecule. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional protein sequence.
By xe2x80x9cspecifically hybridizesxe2x80x9d is meant that a nucleic acid molecule that is capable of hybridizing to a nucleic acid sequence (for example, DNA) at least under low stringency conditions, and preferably under high stringency conditions.
By xe2x80x9cproteinxe2x80x9d is meant any chain of amino acids, including polypeptides, regardless of length or post-translational modification (for example, glycosylation or phosphorylation), including polypeptides.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the nucleic acid molecule (for example, DNA) is positioned adjacent to a sequence which directs transcription of the nucleic acid molecule (for example, a gene expressing a nontranslatable antisense sequence or sense nontranslatable sequence).
By xe2x80x9cexpression control regionxe2x80x9d is meant any minimal sequence sufficient to direct transcription. Included in the invention are promoter and enhancer elements that are sufficient to render promoter-dependent gene expression controllable for cell-, tissue-, or organ-specific gene expression, or elements that are inducible by external signals or agents (for example, light-, pathogen-, wound-, stress- or hormone-inducible elements; or constitutive elements); such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene or engineered into a transgene construct.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence(s) are connected in such a way to permit gene expression when the appropriate molecules (for example, transcriptional activator proteins) are bound to the regulatory sequence(s).
By xe2x80x9cplant cellxe2x80x9d is meant any self-propagating cell bounded by a semi-permeable membrane and containing a plastid. A plant cell, as used herein, is obtained from, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, protoplasts, leaves, roots, shoots, somatic and zygotic embryos, as well as any part of a reproductive or vegetative tissue or organ.
By xe2x80x9cplant componentxe2x80x9d is meant a part, segment, or organ obtained from an intact plant or plant cell. Exemplary plant components include, without limitation, somatic embryos, leaves, fruits, scions and rootstocks.
By xe2x80x9cvineyardxe2x80x9d is meant a plot of land which includes three or more transgenic grapevines or grapevine components which were selected for low level expression of a grape nepovirus coat protein nucleic acid molecule or fragment thereof.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a nucleic acid molecule (for example, a DNA sequence) which is inserted by artifice into a cell and becomes part of the genome of the organism (in either an integrated or extrachromosomal fashion for example, a viral expression construct which includes a subgenomic promoter) which develops from that cell. As used herein, the transgenic organisms are generally transgenic grapevines or grapevine components and the nucleic acid molecule (for example, a transgene) is inserted by artifice into the nuclear or plastidic compartments of the plant cell. Preferably, such transgenic grapevine or grapevine component express at least one sense translatable, sense nontranslatable, or antisense grape nepovirus transcript (for example, a GFLV-CP antisense sequence).
By xe2x80x9ctransgenexe2x80x9d is meant any piece of a nucleic acid molecule (for example, DNA) which is inserted by artifice into a cell, and becomes part of the organism (integrated into the genome or maintained extrachromosomally) which develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9cantisense nucleic acid sequencexe2x80x9d is meant a nucleotide sequence that is complementary to a transcribed RNA. In general, such an antisense sequence will-usually be at least 15 nucleotides, preferably about 15-200 nucleotides, and more preferably 200-2,000 nucleotides in length. The antisense sequence may be complementary to all or a portion of the transcribed RNA nucleotide sequence (for example, a grape nepovirus sequence such as the GFLV-CP antisense constructs described herein or any of the GFLV sequences described in Brandt et al., Arch. Virol. 140: 157-164, 1995; Margis et al., J. Gen. Virol. 74: 1919-1926, 1993; Fuchs et al., J. Gen Virol. 955-962, 1989; Serghini et al., J. Gen. Virol. 71: 1433-1441, 1990; Bardonnet et al., Plant Cell Reports 13: 357-360, 1994; Krastanova et al., Plant Cell Rep. 14:550-554, 1995; Ritzenthaler et al., J. Gen. Virol. 72: 2357-2365, 1991; Mauro et al., Plant Science: 112: 97-106, 1995; and Sanchez et al., Nucleic Acids. Res. 19: 5440, 1992), and, as appreciated by those skilled in the art, the particular site or sites to which the antisense sequence binds as well as the length of the antisense sequence will vary, depending upon the degree of inhibition desired and the uniqueness of the antisense sequence. Preferably, a transcriptional construct expressing a grape nepovirus sequence (for example, a GFLV-CP antisense nucleotide sequence) includes, in the direction of transcription, an expression control region, the sequence coding for the antisense RNA on the sense strand, and a transcriptional termination region. Antisense grape nepovirus sequences (for example, GFLV sequences) may be constructed and expressed as described herein or as described, for example, in van der Krol et al., Gene 72: 45, 1988; Rodermel et al., Cell 55: 673, 1988; Mol et al., FEBS Lett. 268: 427, 1990; Weigel and Nilsson, Nature 377: 495, 1995; Cheung et al., Cell 82: 383, 1995); and U.S. Pat. No. 5,107,065.
By xe2x80x9cincreased resistance to a fanleaf diseasexe2x80x9d is meant a greater level of resistance to a fanleaf disease (for example, any disease caused by a grape nepovirus such as those caused by GFLV, arabis mosaic virus, and the like) in a transgenic grapevine (or grapevine component or cell or seed thereof) than the level of resistance relative to a control grapevine (for example, a non-transgenic grapevine). In preferred embodiments, the level of resistance in a transgenic grapevine is at least 5 to 10% (and preferably 20%, 30%, or 40%) greater than the resistance of a control grapevine. In other preferred embodiments, the level of resistance to fanleaf disease is 50% greater, 60% greater, and more preferably even 75% or 90% greater than a control grapevine; with up to 100% resistance as compared to a control grapevine being most preferred. The level of resistance is measured using conventional methods. For example, the level of resistance to fanleaf disease may be determined by comparing physical features and characteristics (for example, plant height and weight, or by comparing disease symptoms, for example, delayed lesion development, reduced lesion size, leaf wilting and curling, mottling and necrosis of leaves, deformity of canes, number of intemodes, mosiac rings on leaves, and discoloration of cells) of transgenic grapevines. Infectivity of a grape nepovirus (for example, a GFLV or an arabis mosaic virus) can also be monitored using, for example, standard ELISA.
As is discussed above, it has been discovered that the low level expression of a grape nepovirus sense-translatable coat protein gene, as well as an antisense sequence, provides transgenic grapevines with resistance against disease caused by a grape nepovirus. Accordingly, the invention provides a number of important advances and advantages for viticulturists. For example, by selecting transgenic grapevines which express low levels of a recombinant grape nepovirus coat protein gene and thus have increased resistance against grape nepovirus infection, the invention facilitates an effective and economical means for protection against grapevine fanleaf disease and other grape nepovirus-induced diseases. Such protection reduces or minimizes the need for traditional chemical practices (for example, soil fumigation) typically used by viticulturists for controlling the spread of a grape nepovirus and provides protection against these disease-causing pathogens. In addition, because grape plants expressing such grape nepovirus sequences are less vulnerable to grape nepovirus infection and fanleaf disease, the invention further provides for increased production efficiency, as well as for improvements in quality, color, flavor, and yield of grapes. Furthermore, because the invention reduces the necessity for chemical protection against grapevine pathogens, the invention also benefits the environment where the vineyards are planted. The invention may also be used in combination with cultivated rootstocks having resistance to soil-borne nematodes.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.